PROJECT SUMMARY/ABSTRACT ? MASS SPECTROMETRY CORE The Mass Spectrometry Core will support research into the causes of tau toxicity in the context of Frontotemporal Dementia (FTD). The core will apply quantitative proteomics approaches to characterize post- translational modifications (PTMs) and protein-protein interactions (PPIs) of wildtype and FTD-mutants of tau. Although tau mutations and dysregulations are prominent in FTD, little is known about tau function in this disease, which is a common cause of young-onset dementia. A thorough description and exploration of tau- mediated FTD will provide a significant and essential resource for the neurological disorder community. We will comprehensively characterize tau PTMs in different cellular compartments and tissues using a novel platform we have developed that allows for the selective enrichment of tau from human brain tissues and cells. Combined with quantitative mass spectrometry (MS) approaches, we will comprehensively monitor acetylation, phosphorylation, ubiquitylation and methylation sites and determine their regulation in the context of FTD mutations. We will validate FTD mutant regulated PTM sites using a targeted approach called selected reaction monitoring (SRM). In contrast to systematic PTM site mapping which is used for hypothesis generation, SRM allows hypothesis testing by quantifying an a priori selected set of PTM sites in a highly accurate, sensitive, and reproducible fashion across many conditions and larger sample sets. Additionally, we will generate PPI maps for tau in the context of mutations, perturbations, and different cellular localizations. We will use classical affinity purification followed by MS (AP-MS) and novel APEX-based proximity biotinylation to identify tau-interacting proteins mediating aberrant activity and homeostasis in FTD neurons. We will study the interactome associated with both normal and FTD-mutants of tau in autophagosomes, autolysosomes and endosomes, and characterize the mechanisms of how tau is released in an activity-dependent manner as well as study the cellular machinery involved in normal or mutant tau uptake. The Core will interact with the CRISPR Core to functionally validate proteomics data and with the Data Core to integrate datasets and share results with the scientific community.